The following Specific Aims are formulated to address three fundamental issues: (1) how autophagosomes formed at distal terminals acquire motility for transport to the soma for degradation; (2) how endolysosomal trafficking pathways maintain synaptic homeostasis by removing dysfunctional and non-recyclable presynaptic components; and (3) how endosome-lysosome deficits contribute to axonal mitochondrial pathology in spinal motor neurons of fALS-linked mice. Our snapin mice represent one of the few genetic models displaying striking phenotypes in the transport of late endosomes, thus providing us with unique genetic tools for investigating the roles of the autophagy-lysosomal system in neurodegeneration. We established primary motor neuron cultures isolated from adult fALS mice, which provide ideal cell models for delineating transport defects underlying adult-onset pathogenesis in neurodegenerative diseases. Specific Aim 1. Retrograde Transport Regulates Neuronal Autophagy-Lysosomal Function. Late endocytic trafficking, which delivers target materials into lysosomes, is critical for maintaining neuronal degradation capacities via autophagy-lysosomal pathways. Dynein motor-mediated retrograde transport can enhance late endocytic trafficking from distal processes to the soma, where lysosomes are predominantly localized, thus ensuring proper autophagy-lysosomal function. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and endolysosomal trafficking. Snapin acts as a motor adaptor by attaching dynein to late endosomes. Snapin (-/-) neurons exhibit aberrant accumulation of immature lysosomes, impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis, and impaired clearance of autolysosomes. Snapin deficiency leads to axonal degeneration and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes by enhancing the delivery of endosomal cargos to lysosomes. Our studies elucidate a new mechanism coordinating dynein-mediated late endocytic transport and endosomal-lysosomal trafficking. Such a mechanism is critical for maintaining cellular homeostasis essential for neuronal survival. Autophagy-lysosomal dysfunction is one of the cellular defects contributing to the pathogenesis of neurodegenerative diseases associated with accumulation of aggregation-prone proteins and damaged organelles. Specific Aim 2. Endolysosomal Transport and Sorting Regulates Synaptic Activity. Proper regulation of synaptic vesicle (SV) pool size is critical to maintain synaptic activity. Because early endosomes represent crossroads between local SV recycling and the endolysosomal degradation system, this raises a fundamental question: are SVs sorted toward endolysosomal pathways for degradation? To address this question, we applied snapin dominant-negative mutants as molecular tools combined with dual-channel time-lapse imaging in live cortical neurons. Our study reveals that dynein-driven late endosome transport regulates the SV pool size. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin-/- phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. SV components are co-transported with late endosomes along axons. Therefore, our study provides new mechanistic insights into maintaining and regulating SV pool size through snapin-mediated endosomal trafficking and sorting. Specific Aim 3. Mechanism of Autophagosome Transport in Axons. Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process over a neurons lifetime. Autophagy undergoes stepwise maturation: bulk cytoplasmic components and organelles are engulfed within double-membrane organelles termed autophagosomes, followed by fusion with late endosomes (LEs) into amphisomes, or fusion with lysosomes into autolysosomes for degradation in the soma. However, it is unknown how nascent autophagosomes in distal axons acquire their retrograde motility. We reveal a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. LE-loaded dynein-snapin motor-adaptor complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor-sharing mechanism enables neurons to maintain effective autophagic clearance in the soma, thus reducing autophagic stress in axons. Blocking dynein recruitment to LEs by disrupting dynein-snapin coupling impairs the movement of amphisomes toward the cell body. Reducing the ability of autophagosomes to fuse with LEs results in aberrant accumulation of immobile AVs in axonal terminals. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases. Specific Aim 4. Autophagy-Lysosomal Deficits in ALS-linked Early Pathology. One pathological hallmark in fALS-linked motor neurons (MNs) is axonal accumulation of autophagic vacuoles (AVs), thus raising a fundamental question as to whether reduced autophagic clearance due to an impaired lysosomal system contributes to autophagic stress and axonal degeneration. We recently revealed progressive lysosomal deficits in spinal MNs beginning at early asymptomatic stages in fALS-linked mice expressing the human SOD1G93A protein. Such deficits impair the degradation of AVs engulfing damaged mitochondria from distal axons. These early pathological changes are attributable to mutant hSOD1, which interferes with dynein-driven endolysosomal trafficking. Elucidation of this pathological mechanism is broadly relevant, because autophagy-lysosomal deficits are associated with several major neurodegenerative diseases. Therefore, enhancing lysosome function, rather than autophagy induction, is an alternative and promising therapeutic strategy for ALS-linked clinical trials. Specific Aim 5. Anterograde Axonal Transport Regulates Synaptic Formation and Plasticity. The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity and require the targeted delivery of newly synthesized synaptic cargoes from the soma to synapses. Our previous studies established that syntabulin is an adaptor capable of linking KIF5 motor and synaptic protein cargoes. Syntabulin-KIF5 coupling mediates axonal transport of synaptic components essential for presynaptic assembly and maintenance. Syntabulin loss-of-function blocks formation of new presynaptic terminals in developing neurons. Our studies establish that kinesin-mediated axonal transport is one mechanism underlying activity-dependent presynaptic plasticity. Conditional syntabulin knockout mice have been recently generated in the lab. We will use this mouse line to determine whether deficiency in syntabulin-mediated synaptic cargo transport has any impact on synapse formation, maintenance and plasticity during health brain development and in neurological and mental disorders.